Abstract The continuous epidemic of Middle East respiratory syndrome (MERS) caused by MERS-coronavirus (MERS- CoV) has posed a threat to global public health, calling for the development of innovative and effective intervention strategies. Our previous studies have demonstrated that the receptor-binding domain (RBD) in the MERS-CoV spike (S) protein, which mediates virus binding to the receptor dipeptidyl peptidase 4 (DPP4), contains a critical neutralizing domain (CND), serving as an important target for the development of vaccines and neutralizing antibodies. A number of RBD-targeting neutralizing monoclonal antibodies (mAbs) with potent neutralizing activity and/or protection against MERS-CoV infection have been reported. In spite of their superior safety and strong specificity, these conventional antibodies have large size (e.g., 160 kDa for IgG) and complicated structures. These characteristics result in instability and limit rapid, large-scale production. Different from conventional antibodies, variable domains of camelid heavy chain-only antibody (VHH), or nanobody (Nb), has small molecular size (~15 kDa), strong thermal stability, high production yield and other good druggabilities, making it particularly suitable for rapid and large-scale production as an antiviral drug with affordable price to meet healthcare needs during an outbreak. We have previously developed several effective RBD-specific mouse and human neutralizing mAbs and identified three RBD-targeting neutralizing Nbs. Based on our experience and preliminary data, we hypothesize that a novel, stable, and effective RBD-specific neutralizing Nb can be developed to combat MERS. The specific aims of this proposal are: 1) to generate humanized MERS-CoV RBD-specific neutralizing Nbs with extended half-lives; 2) to characterize MERS-CoV RBD-specific humanized Nbs; and 3) to evaluate the in vitro and in vivo efficacy of RBD-specific humanized Nbs against MERS-CoV infection. The long-term goal of the proposed study is to develop a novel nanobody with good druggability for the prevention and treatment of MERS-CoV infection. Similar approaches can be applied to develop prophylactic and therapeutic agents against other class I enveloped viruses with epidemic/pandemic potential.